High affinity TCR proteins and methods

ABSTRACT

T cell receptors (TCRs) that have higher affinity for a ligand than wild type TCRs are provided. These high affinity TCRs are formed by mutagenizing a T cell receptor protein coding sequence to generate a variegated population of mutants of the T cell receptor protein coding sequence; transforming the T cell receptor mutant coding sequence into yeast cells; inducing expression of the T cell receptor mutant coding sequence on the surface of yeast cells; and selecting those cells expressing T cell receptor mutants that have higher affinity for the peptide/MHC ligand than the wild type T cell receptor protein. The high affinity TCRs can be used in place of an antibody or single chain antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/009,388, filed Jan. 20, 1998. This application claims thebenefit of U.S. Provisional Application No. 60/169,179, filed Dec. 6,1999.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

[0002] This invention was made, at least in part, with funding from theNational Institutes of Health. Accordingly, the United States Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] The field of the present invention is molecular biology, inparticular, as it is related to combinatorial libraries of immune cellreceptors displayed on the cell surface of a recombinant host cell. Morespecifically, the present invention relates to a library of highaffinity T cell receptor proteins displayed on the surfaces ofrecombinant yeast cells, to soluble high affinity TCR receptor proteins,to high affinity TCR proteins selected for high affinity binding toparticular peptide/MHC pairs, to high affinity TCR proteins selected forbinding to a particular antigen in the absence of an MHC determinant,and to the use of the selected high affinity TCR derivatives indiagnostic methods and imaging assays, among other applications.

[0004] T cell receptors (TCRs) and antibodies have evolved to recognizedifferent classes of ligands. Antibodies function as membrane-bound andsoluble proteins that bind to soluble antigens, whereas in nature, TCRsfunction only as membrane-bound molecules that bind to cell-associatedpeptide/MHC antigens. All of the energy of the antibody:antigeninteraction focuses on the foreign antigen, whereas a substantialfraction of the energy of the TCR peptide/MHC interaction seems to bedirected at the self-MHC molecule [Manning et al. (1998) Immunity8:413:425]. In addition, antibodies can have ligand-binding affinitiesthat are orders of magnitude higher than those of TCRs, largely becauseof the processes of somatic mutation and affinity maturation. In theirnormal cellular context, TCRs do not undergo somatic mutation, and theprocesses of thymic selection seem to operate by maintaining a narrowwindow of affinities [Alam et al. (1996) Nature 381:616-620]. Theassociation of TCRs at the cell surface with the accessory molecules CD4or CD8 also may influence the functional affinity of TCRs [Garcia et al.(1996) Nature 384:577-581]. Despite these differences, thethree-dimensional structures of the two proteins are remarkably similar,with the hypervariable regions forming loops on a single face of themolecule that contacts the antigen.

[0005] Based on their structural similarities, it is somewhat surprisingthat there have been significant differences in the success of producingsoluble and surface-displayed forms of the extracellular domains of TCRsand antibodies in heterologous expression systems. Many antibodies havenow been expressed at high yield and solubility as either intact orFab-fragment forms or as single-chain (sc) fragment-variable (Fv)proteins. In addition, there are numerous antigen-binding Fv fragmentsthat have been isolated de novo and/or improved through the use ofphage-display technology and, more recently, with yeast-displaytechnology [Boder and Wittrup (1997) Nat. Biotechnol. 15:553-557; Kiekeet al. (1997) Prot. Eng. 10:1303-1310]. These expression systems forantibody fragments have been key in structural studies and in the designof diagnostic and therapeutic antibodies.

[0006] In contrast, the three-dimensional structures of a few TCRmolecules were determined only after considerable effort on theexpression of soluble, properly folded TCRs [Bentley and Mariuzza (1996)Ann. Rev. Immunol. 14:563-590]. One of the difficulties in exploring thebasis of differences between Fab and TCR has been that the extensivesequence diversity in antibody and TCR variable (V) regions complicatesefforts to discern what features of the V regions are important forfunctions other than antigen binding (e.g., V region pairing andassociation kinetics, stability, and folding). There have beenrelatively few studies that have compared the V regions of TCRs andantibodies in terms of these properties.

[0007] Nevertheless, the TCR from the mouse T cell clone 2C has now beenexpressed as an sc V_(α)V_(β)(scTCR) in Escherichia coli [Soo Hoo et al.(1992) Proc. Natl. Acad. Sci. USA 89:4759-4763], as a lipid-linkedV_(α)C_(α)V_(β)C_(β) dimer from myeloma cells [Slanetz and Bothwell(1991) Eur. J. Immunol. 21:179-183], and as a secreted V_(α)C_(α)V_(βC)_(β) dimer from insect cells [Garcia et al. (1996) Science 274:209-219].The 2C scTCR had relatively low solubility compared with most scFv,although its solubility is increased about 10-fold by fusion at theamino terminus to thioredoxin [Schodin et al. (1996) Molec. Immunol.33:819-829]. The difficulty in generating soluble, properly foldedV_(α)V_(β) domains has extended to other TCRs [Udaka et al. (1993)supra; Sykulev et al. (1994) supra; Manning et al. (1998) supra]. Themolecular explanation for the apparent differences between TCR and Fv ineither solubility or surface-display capability has not been exploredadequately. It has been shown that the 2C scTCR can be expressed in ayeast surface-display system after the selection, from a random library,of specific single-site mutations at the V_(α)/V_(β) interface or in aregion of the V_(β) framework suspected to interact with the CD3_(ε)signal-transduction sub-unit. These mutations, several of which arefound naturally in antibody V regions, reflect the significance of thesepositions in the TCR and provide a basis for further engineering ofTCR-binding properties.

SUMMARY OF THE INVENTION

[0008] The invention provides a combinatorial library of immune T cellreceptor polypeptides displayed on the surfaces of recombinant hostcells, for example, yeast cells, desirably Saccharomyces cerevisiae.From such a library can be isolated high affinity TCR polypeptides(those that exhibit higher affinity than wild type for the cognateligand: a complex of peptide bound to a protein of the majorhistocompatibility complex, pMHC). Desirably, the affinity of the TCRpeptide for the pMHC is reflected in a dissociation constant of fromabout 10⁷ to about 10¹⁰, e.g., as measured by methods known to the art.A DNA library comprising nucleic acids encoding soluble high affinityTCRs, wherein said TCRs are made by the method of mutagenizing a TCR tocreate mutant TCR coding sequences; transforming DNA comprising themutant TCR coding sequences for mutant TCRs into yeast cells; inducingexpression of the mutant TCR coding sequences such that the mutant TCRsare displayed on the surface of yeast cells; contacting the yeast cellswith a fluorescent label which binds to the peptide/MHC ligand toproduce selected yeast cells; and isolating the yeast cells showing thehighest fluorescence is provided. Also provided is a library of T cellreceptor proteins displayed on the surface of yeast cells which havehigher affinity for the peptide/MHC ligand than the wild type T cellreceptor protein, wherein said library is formed by mutagenizing a Tcell receptor protein coding sequence to generate a variegatedpopulation of mutants of the T cell receptor protein coding sequence;transforming the T cell receptor mutant coding sequence into yeastcells; inducing expression of the T cell receptor mutant coding sequenceon the surface of yeast cells; and selecting those cells expressing Tcell receptor mutants that have higher affinity for the peptide/MHCligand than the wild type T cell receptor protein.

[0009] The present invention further provides TCR proteins (incell-bound or in soluble form) that exhibit high affinity binding forthe cognate ligand. In the present invention the ligand bound by the TCRprotein can be a peptide/MHC complex or because of the selectionprocess, desirably an iterated selection process, it can be a ligandwhich does not include an MHC component, such as a superantigen. Thisligand can be a peptide, a protein, a carbohydrate moiety, or a lipidmoiety, among others. These soluble high affinity TCRs may be made bythe method comprising: mutagenizing a TCR to create mutant TCR codingsequences; transforming DNA comprising the mutant TCR coding sequencesfor mutant TCRs into yeast cells; inducing expression of the mutant TCRcoding sequences such that the mutant TCRs are displayed on the surfaceof yeast cells; contacting the yeast cells with a fluorescent labelwhich binds to the peptide/MHC ligand to produce selected yeast cells;and isolating the yeast cells showing the highest fluorescence. Thesoluble high affinity TCRs are preferably isolated by yeast display.

[0010] The present invention further provides methods for detecting thecognate ligand of a high affinity TCR protein, said methods comprisingthe step of binding the high affinity TCR protein with the cognateligand, where the high affinity TCR protein is detectably labeled orwhere there is a secondary detectable protein added, such as an antibodyspecific for the TCR in a region other than the region which binds thecognate ligand. A preferred method for using high affinity TCRs toidentify ligands comprises: labeling high affinity TCRs with adetectable label; contacting said labeled TCRs with ligands; identifyingthe ligand with which the labeled TCR is bound. Preferably the ligandsare those peptide/MHC ligands to which the TCR binds with higheraffinity than the wild type TCR. Methods of identifying the ligand areknown to one of ordinary skill in the art. Suitable labels allowing fordetection of the TCR protein, directly or indirectly, include but arenot limited to fluorescent compounds, chemiluminescent compounds,radioisotopes, chromophores, and others.

[0011] The high affinity TCR protein can be used in the laboratory as atool for qualitative and quantitative measurements of a target ligand,in medical, veterinary or plant diagnostic setting or for tissue orplant material identification. Similarly, the high affinity TCRs of thepresent invention can be used as reagents for detecting and/orquantitating a target material or ligand. Also provided is a method ofusing high affinity TCRs to bind to a selected peptide/MHC ligandcomprising: labeling said high affinity TCRs with a label that binds tothe selected peptide/MHC ligand; contacting said labeled high affinityTCRs with cells containing MHC molecules. The high affinity protein ofthe present invention, where it specifically binds to a tumor cellantigen with high affinity and specificity can be used in diagnostictests for the particular type of cancer or it can be used in an organismin imaging tests to locate and/or estimate size and number of tumors inan organism, preferably a mammal, and also preferably a human. Methodsprovided for using high affinity TCRs that bind to pMHCs for diagnostictests comprise: labeling the high affinity TCR with a detectable label;contacting said high affinity TCR with cells containing the ligand towhich the high affinity TCR has high affinity for; and detecting thelabel. In the method, the label may be chosen to bind to specificpeptide/MHC ligands, whereby cells that express specific peptide/MHCligands are targeted. Preferred methods for using high affinity TCRs asdiagnostic probes for specific peptide/MHC molecules on surfaces ofcells comprise: labeling high affinity TCRs with a detectable label thatbinds to specific peptide/MHC ligands; contacting said TCRs with cells;and detecting said label. The detectable label chosen for use depends onthe particular use, and the choice of a suitable label is well withinthe ordinary skill of one in the relevant art. In general, the TCRproteins selected for high affinity binding to a ligand of interest canbe used in methods in which antibodies specific for the ligand can beused, with procedural modifications made for the TCR vs. antibodyprotein, such modifications being known in the art.

[0012] The high affinity TCR, desirably a soluble single chain (sc) TCR,can be used to block autoimmune destruction of cells or tissues inautoimmune disease, where the site recognized by the cytotoxiclymphocytes on the surface of the target cell is the same as the sitebound by the high affinity TCR. Preferred methods for blockingautoimmune destruction of cells comprise contacting TCRs with highaffinity for the site recognized by the T lymphocytes on the surface ofa target cell with cells, whereby the autoimmune destruction of cells isblocked.

[0013] A soluble, high affinity scTCR can be coupled to a therapeuticcompound (e.g., an anticancer compound, a therapeutic radionuclide or acytoxic protein) where the cognate ligand of the sc TCR is a neoplasticcell surface marker. Alternatively, the binding specificity of the highaffinity soluble sc TCR can be a pathogen infected target cell (such asvirus-, bacteria- or protozoan-infected) and a toxic molecule can becoupled so that the target cell can be eliminated without furtherreplication of the infective agent. Provided methods of using highaffinity TCRs to inactivate pathogens comprise: binding a molecule whichis toxic to the pathogen to the high affinity TCR; and contacting saidTCR with cells that express said pathogen. “Toxic” means that thepathogen prevents or inhibits replication of the pathogen.

[0014] Also provided are methods for using high affinity TCRs to treatdisease comprising: coupling a TCR having a high affinity for aneoplastic cell surface marker with a therapeutic compound; andcontacting said TCR with cells. Any therapeutic compound that is usefulin slowing the progress of the disease that can be coupled with the TCRmay be used. Methods of coupling the therapeutic compound with the TCRare known in the art.

[0015] Also provided is a method for cloning the gene for a highaffinity TCR mutant into a system that allows expression of the mutanton the surface of T cells comprising: mutating TCRs to create highaffinity TCR mutants; cloning said TCR mutants into a vector;transfecting the vector into T cells; expressing the high affinity TCRmutant on the surface of T cells. This method may further comprise:selecting those T cells that are activated to a greater extent thanother T cells by a peptide/MHC ligand. The transfected/infected T cellsmay be used for recognition of selected peptide-bearing MHC cells. Thesetransfected/infected T cells are useful in treating disease in patientswhere T cells from a patient are removed and transformed with the vectorthat expresses the high affinity TCR mutants and returned to the patientwhere they are activated to a greater extent by a peptide/MHC ligandthan the patient's wild type T cells.

[0016] A soluble, high affinity TCR molecule can be used in place of anantibody or single chain antibody for most applications, as will bereadily apparent to one of skill in the relevant arts.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1: Flow cytometric analysis of yeast cells that expresswild-type and mutant 2C TCR on their surfaces. Yeast cells displayingwild-type (T7) and mutant (qL2, qL7) scTCR were stained with anti-Vβ8antibody F23.2 (120 nM), the specific alloantigenic peptide-MHC,QL9/L^(d)/Ig (40 nM), or a null peptide MCMV(SEQ ID NO:1)L^(d)/Ig (40nM). Binding was detected by FITC-conjugated goat anti-mouse IgG F(ab′)₂and analyzed by flow cytometry. The negative population (e.g. seen withF23.2 staining) has been observed for all yeast displayed-proteins andis thought to be due to cells at a stage of growth or induction that areincapable of expressing surface fusion protein (Kieke et al. (1999)Proc. Natl. Acad. Sci. USA 96:5651-5656; Boder and Wittrup (1997) Nat.Biotech. 15:553-557; Kieke et al. (1997) Protein Engineering10:1303-1310).

[0018]FIG. 2: Fine specificity analysis of mutant scTCR binding todifferent QL9 variant peptides bound to L^(d). The original T cell clone2C and various yeast clones were analyzed by flow cytometry for bindingto L^(d)/Ig dimers loaded with wild type QL9 (P5F), position 5 variantsof QL9 (P5Y, P5H, P5E) or MCMV (SEQ ID NO:1). Binding was detected withFITC-labeled goat anti-mouse IgG. Relative fluorescence was measured bytwo different approaches. For T cell clone 2C, the binding of thevarious peptide/L^(d) Ig dimers was adjusted relative to the QL9/L^(d)staining (MFU_(pMHC)/MFU_(QL)9-Ld). For yeast cells, the binding of eachpeptide/L^(d) dimer was adjusted relative to binding by the anti-Vβ8antibody F23.2 (MFU_(pMHC)/MFU_(F23.2)). The latter allowed differentmutants to be compared relative to each other for binding to the wildtype QL9/L^(d).

[0019]FIG. 3: QL9/L^(d) binding by soluble scTCRs. T2-L^(d) cells loadedwith QL9 were incubated with ¹²⁵I-labeled anti-L^(d) Fab fragments(30-5-7) and various concentrations of unlabeled Fab (♦), scTCR-T7 (▪),or mutant scTCR-qL2 (). Bound and unbound ¹²⁵I 30-5-7 Fab fragmentswere separated by centrifugation through olive oil/dibutyl phthalate.Binding of ¹²⁵I-labeled anti-L^(d) Fab fragments to T2-L^(d) cellsloaded with the control peptide MCMV (SEQ ID NO:1) was not inhibitedeven at the highest concentrations of scTCRs (data not shown).

[0020]FIG. 4: Flow cytometric analysis of the binding of scTCR/biotin tocell surface peptide/MHC. Peptide-loaded T2-L^(d) cells were incubatedwith biotinylated qL2 scTCR (˜0.3 μM) or T7 scTCR (˜1.6 μM) scTCRfollowed by streptavidin-PE and analyzed by flow cytometry. FIG. 4A:Flow cytometry histograms of T2-L^(d) cells loaded with QL9 (unshaded),p2Ca (light shade), or MCMV (SEQ ID NO:1) (dark shade) and stained withqL2 scTCR/biotin. FIG. 4B: Mean fluorescent units (MFU) of T2-L^(d)cells loaded with QL9, p2Ca, or MCMV (SEQ ID NO:1) and stained witheither secondary SA-PE only, T7 scTCR/biotin+SA-PE, or qL2scTCR/biotin+SA-PE. FIG. 4C: A soluble, high affinity form of mutant qL2expressed from insect cells can detect very low concentrations of apeptide-MHC complex. Ld complexes were up-regulated on the surface ofT2-Ld cells (3×10⁶/ml) by incubation with various concentrations of QL9peptide at 37° C. for 1.5 hr. Approximately 2×10⁵ cells were stained for30 min on ice with TCRs derived from transfected Drosophila melanogaster(insect) SC2 cells (Garcia, K. C., et al. (1997) Proc Natl Acad Sci USA94(25), 13838-13843). Cells were then washed and stained withbiotin-labeled anti-Vb IgG (F23.1) followed by streptavidin-PE andanalyzed by flow cytometry.

[0021]FIG. 5: Flow cytometry histograms of yeast displaying a mutantscTCR (called 3SQ2) stained with biotinylated peptide/MHC complexes,OVA/K^(b), dEV8/K^(b) or SIYR (SEQ ID NO:2)/K^(b), followed bystreptavidin-PE. As a positive control for the presence of scTCR, yeastwere stained with the Vβ-specific Ig, F23.2 followed by FITCgoat-anti-mouse F(ab′)₂.

[0022]FIG. 6: T2-K^(b) tumor cells were incubated with specific peptides(OVA, dEV8 or SIYR (SEQ ID NO:2)) and analyzed by flow cytometrystaining with biotinylated soluble scTCR, 3SQ2 followed bystreptavidin-PE. As a positive control for the presence of K^(b),T2-K^(b) cells were stained with biotinylated antibody B8.24.3, whichrecognizes K^(b) irrespective of the bound peptide.

[0023]FIG. 7: After multiple rounds of sorting with dEV8/K^(b), theyeast VαCDR3 library was stained with biotinylated dEV8/K^(b) followedby streptavidin-PE and analyzed by flow cytometry.

[0024]FIG. 8: Flow cytometry histograms of yeast displaying a mutantscTCR (called 4d1) stained with biotinylated peptide/MHC complexes,OVA/K^(b) or dEV8/K^(b) followed by staining with biotinylatedstreptavidin-PE. As positive controls the yeast were analyzed for thepresence of scTCR Vβ with F23.2 Ig and for epitope tags with ananti-6His antibody or the anti-HA Ig, 12CA5.

[0025]FIG. 9: T cells transfected with the mutant T cell receptor qL2can recognize and be stimulated by target cells that express thepeptide-MHC at low concentrations. T-cell hybridoma cell line 58-/-(Letourneur, F., and B. Malissen. (1989) Eur J Immunol 19(12), 2269-74)was transfected with the wild-type (2C) or mutant (qL2) TCRs and 7.5×10⁴transfected cells/well were incubated at 37° C. with T2-Ld cells(7.5×10⁴/well) in the presence of QL9 peptide. After ˜30 hrs,supernatants were collected and assayed for IL-2 released by the Tcells: Supernatants were incubated with the IL-2 dependent cell line,HT2 (5×10³/well) for 18 hrs at 37° C. Proliferation of HT2 cells wasmeasured by the incorporation of 3 [H] thymidine. Mean CPM representsthe average of triplicate wells. No IL-2 was released in the absence ofthe QL9 peptide (data not shown).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given to such terms,the following definitions are provided.

[0027] A coding sequence is the part of a gene or cDNA which codes forthe amino acid sequence of a protein, or for a functional RNA such as atRNA or rRNA.

[0028] Complement or complementary sequence means a sequence ofnucleotides which forms a hydrogen-bonded duplex with another sequenceof nucleotides according to Watson-Crick base-pairing rules. Forexample, the complementary base sequence for 5′-AAGGCT-3' is3′-TTCCGA-5′.

[0029] Downstream means on the 3′ side of any site in DNA or RNA.

[0030] Expression refers to the transcription of a gene into structuralRNA (rRNA, tRNA) or messenger RNA (mRNA) and subsequent translation of amRNA into a protein.

[0031] An amino acid sequence that is functionally equivalent to aspecifically exemplified TCR sequence is an amino acid sequence that hasbeen modified by single or multiple amino acid substitutions, byaddition and/or deletion of amino acids, or where one or more aminoacids have been chemically modified, but which nevertheless retains thebinding specificity and high affinity binding activity of a cell-boundor a soluble TCR protein of the present invention. Functionallyequivalent nucleotide sequences are those that encode polypeptideshaving substantially the same biological activity as a specificallyexemplified cell-bound or soluble TCR protein. In the context of thepresent invention, a soluble TCR protein lacks the portions of a nativecell-bound TCR and is stable in solution (i.e., it does not generallyaggregate in solution when handled as described herein and understandard conditions for protein solutions).

[0032] Two nucleic acid sequences are heterologous to one another if thesequences are derived from separate organisms, whether or not suchorganisms are of different species, as long as the sequences do notnaturally occur together in the same arrangement in the same organism.

[0033] Homology refers to the extent of identity between two nucleotideor amino acid sequences.

[0034] Isolated means altered by the hand of man from the natural state.If an “isolated” composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not isolated, but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is isolated, as theterm is employed herein.

[0035] A linker region is an amino acid sequence that operably links twofunctional or structural domains of a protein.

[0036] A nucleic acid construct is a nucleic acid molecule which isisolated from a naturally occurring gene or which has been modified tocontain segments of nucleic acid which are combined and juxtaposed in amanner which would not otherwise exist in nature.

[0037] Nucleic acid molecule means a single- or double-stranded linearpolynucleotide containing either deoxyribonucleotides or ribonucleotidesthat are linked by 3′-5′-phosphodiester bonds.

[0038] Two DNA sequences are operably linked if the nature of thelinkage does not interfere with the ability of the sequences to effecttheir normal functions relative to each other. For instance, a promoterregion would be operably linked to a coding sequence if the promoterwere capable of effecting transcription of that coding sequence.

[0039] A polypeptide is a linear polymer of amino acids that are linkedby peptide bonds.

[0040] Promoter means a cis-acting DNA sequence, generally 80-120 basepairs long and located upstream of the initiation site of a gene, towhich RNA polymerase may bind and initiate correct transcription. Therecan be associated additional transcription regulatory sequences whichprovide on/off regulation of transcription and/or which enhance(increase) expression of the downstream coding sequence.

[0041] A recombinant nucleic acid molecule, for instance a recombinantDNA molecule, is a novel nucleic acid sequence formed in vitro throughthe ligation of two or more nonhomologous DNA molecules (for example arecombinant plasmid containing one or more inserts of foreign DNA clonedinto at least one cloning site).

[0042] Transformation means the directed modification of the genome of acell by the external application of purified recombinant DNA fromanother cell of different genotype, leading to its uptake andintegration into the subject cell's genome. In bacteria, the recombinantDNA is not typically integrated into the bacterial chromosome, butinstead replicates autonomously as a plasmid.

[0043] Upstream means on the 5′ side of any site in DNA or RNA.

[0044] A vector is a nucleic acid molecule that is able to replicateautonomously in a host cell and can accept foreign DNA. A vector carriesits own origin of replication, one or more unique recognition sites forrestriction endonucleases which can be used for the insertion of foreignDNA, and usually selectable markers such as genes coding for antibioticresistance, and often recognition sequences (e.g. promoter) for theexpression of the inserted DNA. Common vectors include plasmid vectorsand phage vectors.

[0045] High affinity T cell receptor (TCR) means an engineered TCR withstronger binding to a target ligand than the wild type TCR.

[0046] T cells recognize a foreign peptide bound to the MHC productthrough the αβ heterodimeric T cell receptor (TCR). The TCR repertoirehas extensive diversity created by the same gene rearrangementmechanisms used in antibody heavy and light chain genes [Tonegawa, S.(1988) Biosci. Rep. 8:3-26]. Most of the diversity is generated at thejunctions of variable (V) and joining (J) (or diversity, D) regions thatencode the complementarity determining region 3 (CDR3) of the α and βchains [Davis and Bjorkman (1988) Nature 334:395-402]. However, TCRs donot undergo somatic point mutations as do antibodies and, perhaps notcoincidentally. TCRs also do not undergo the same extent of affinitymaturation as antibodies. TCRs as they occur in nature appear to haveaffinities that range from 10⁵ to 10⁶ M⁻¹ whereas antibodies typicallyhave affinities that range from 10⁵ to 10⁹ M⁻¹ [Davis et al. (1998)Annu. Rev. Immunol. 16:523-544; Eisen et al. (1996) Adv. Protein Chem.49:1-56]. While the absence of somatic mutation in TCRs may beassociated with lower affinities, it has also been argued that there isnot a selective advantage for a TCR to have higher affinity. In fact,the serial-triggering [Valitutti et al. (1995) Nature 375:148-151] andkinetic proofreading [Rabinowitz et al. (1996) Proc. Natl. Acad. Sci.USA 93:1401 - 1405] models of T cell activation both suggest that longeroff-rates (associated with higher affinity) would be detrimental to thesignaling process. It is also possible that higher affinity TCRs mightnot maintain the peptide specificity required for T cell responses. Forexample, peptides bound within the MHC groove display limited accessiblesurface [Bjorkman, P. J. (1997) Cell 89:167-170], which may in turnlimit the amount of energy that can be generated in the interaction. Onthe other hand, raising the affinity of a TCR by directing the energytoward the MHC helices would presumably lead to thymic deletion duringnegative selection [Bevan, M. J. (1997) Immunity 7:175-178].

[0047] We show that there is not an inherent structural property orgenetic limitation on higher affinity of T cell receptor proteins.Higher affinity TCR variants were generated in the absence of in vivoselection pressures by using yeast display combinatorial technology andTCR mutants (e.g., Vα and Vβ CDR3 mutants). Mutants selected forrelatively strong binding to the target ligand (a particular p/MHCcomplex) can have greater than 100-fold higher affinity, i.e., a K_(d)of about 10 nM for the p/MHC, and these mutants retained a high degreeof peptide specificity. A strong preference for TCR proteins withconserved CDR3 motifs that were rich in proline or glycine were alsoevident. A soluble monomeric form of a high affinity TCR was capable ofdirectly detecting p/MHC complexes on antigen-presenting cells. Thesefindings prove that affinity maturation of TCRs is possible, at least invitro. Thus, engineered TCR proteins can be used for targeting specificligands, including particular p/MHC complexes and peptides, proteins orother ligands in the absence of a MHC component.

[0048] To examine if it is possible to generate higher affinity TCR thatretain peptide specificity, we subjected a characterized TCR to aprocess of directed in vitro evolution. Phage display technology[Clackson et al. (1991) Nature 352:624-628] has not yet provensuccessful in the engineering of single-chain TCRs (scTCRs,Vβ-linker-Vα), despite the extensive structural similarity betweenantibody and TCR V regions. However, we recently showed that a scTCRcould be displayed on the surface of yeast [Kieke et al. (1999) Proc.Natl. Acad. Sci. USA 96:5651-5656], in a system that has provensuccessful in antibody engineering [Boder and Wittrup (1997) supra;Kieke et al. (1997) supra]. A temperature-stabilized variant (called T7)[Shusta et al. (1999) J. Mol. Biol. 292:949-956] of the scTCR from theCTL clone 2C was used in the present study. CTL clone 2C recognizes thealloantigen L^(d) with a bound octamer peptide called p2Ca, derived fromthe enzyme 2-oxoglutarate dehydrogenase [Udaka et al. (1993) Proc. Natl.Acad. Sci. USA 90:11272-11276]. The nonameric variant QL9 is alsorecognized by CTL 2C, but with 10-fold higher affinity by the 2C TCR[Sykulev et al. (1994) Proc. Natl. Acad. Sci. USA 91:11487-11491].Alanine scanning mutagenesis shows that the CDR3α loop contributedminimal energy to the binding interaction [Manning (1998) supra], eventhough structural studies have shown that CDR3α of the 2C TCR is nearthe peptide and it undergoes a conformational change in order toaccommodate the pMHC complex [Garcia (1998) Science 279:1166-1172].Thus, we focused our mutagenesis efforts on the five residues that formthe tip of CDR3α.

[0049] A library of 10⁵ independent TCR-CDR3α yeast mutants wassubjected to selection by flow cytometry with a fluorescently-labeledQL9/L^(d) ligand [Dal Porto et al. (1993) Proc. Natl. Acad. Sci. USA90:6671-6675]. After four rounds of sorting and growth, fifteendifferent yeast colonies were examined for their ability to bind theligand, in comparison to the scTCR variant T7, which bears the wt CRD3αsequence (FIG. 1). The anti-Vβ8.2 antibody F23.2 which recognizesresidues in the CDR1 and CDR2 regions of the protein was used as acontrol to show that wt scTCR-T7 and scTCR mutants (qL2 and qL7 in FIG.1 and others) each had approximately equivalent surface levels of thescTCR (FIG. 1). In contrast, the soluble QL9/L^(d) ligand bound verywell to each mutant yeast clone but not to wt scTCR-T7. The MCMV (SEQ IDNO:1)/L^(d) complex, which is not recognized by CTL clone 2C, did notbind to the scTCR mutants or to the wt scTCR-T7, indicating that thescTCR mutants retained peptide specificity. The relative affinities ofthe mutant TCR also appeared to vary among clones, based on differencesin signals observed with the QL9/L^(d) ligand at constantconcentrations.

[0050] The CDR3α sequences of the fifteen mutants all differed from thestarting 2C TCR sequence (Table 1). Comparison by a BLAST alignmentalgorithm aligned the sequences into two motifs. One motif containedglycine in the middle of the 5 residue stretch whereas the other motifcontained three tandem prolines. Evidence that all three prolines areimportant in generating the highest affinity site is suggested byresults with mutant q3r. Mutant q3r contained only two of the threeprolines and exhibited reduced binding compared to the triple-prolinemutants. The glycine-containing mutants appeared to have preferences forpositive-charged residues among the two residues to the carboxy side(7/9) and aromatic and/or positive-charged residues among the tworesidues to the amino side (4/9 and 5/9). Without wishing to be bound bytheory, it is believed that the selection for a glycine residue atposition 102 in the motif indicates that the CDR3α loop requiresconformational flexibility around this residue in order to achieveincreased affinity. This is consistent with the large (6 Å)conformational difference observed between the CDR3α loops of theliganded and unliganded TCR [Garcia et al. (1998) supra]. It is alsointeresting to note that glycine is the most common residue at the V(D)Jjunctions of antibodies and that the presence of a glycine has recentlybeen associated with increased affinity in the response to the(4-hydroxy-3-nitrophenyl) acetyl hapten [Furukawa et al. (1999) Immunity11:329-338].

[0051] In contrast to the isolates that contain glycine, the selectionfor a proline-rich sequence at the tip of the CDR3α loop is believed,without wishing to be bound by any particular theory, to indicate thatthese TCR molecules exhibit a more rigid conformation that confershigher affinity. The X-ray crystallographic structures of a germ lineantibody of low affinity compared to its affinity-matured derivativeshowed that the high affinity state may have been due to thestabilization of the antibody in a configuration that accommodated thehapten [Wedemayer et al. (1997) Science 276:1665-1669]. Similarly, theNMR solution structure of a scTCR that may be analogous to the germlineantibody showed that the CDR3α and β loops both exhibited significantmobility [Hare et al. (1999) Nat. Struct. Biol. 6:574-581]. Recentthermodynamic studies of TCR:pMHC interactions have also suggested theimportance of conformational changes in binding [Willcox et al. (1999)Immunity 10:357-365; Boniface et al. (1999) Proc. Natl. Acad. Sci. USA96:11446-11451]. Structural and thermodynamic studies of the TCR mutantsdiscussed herein allowed us to examine if the two CDR3α motifs (Glyversus Pro-rich) differ in the mechanism by which they confer higheraffinity.

[0052] Although the scTCR mutants did not bind the null (irrelevant)peptide/L^(d) complex MCMV (SEQ ID NO:1)/L^(d), it remained possiblethat the increase in affinity was accompanied by a change in finespecificity. To examine this question, we used QL9 position 5 (Phe)peptide variants which have been shown previously to exhibit significantdifferences in their binding affinity for the wt 2C TCR [Schlueter(1996) J. Immunol. 157:4478-4485]. The binding of these pMHC to variousTCR mutants on the yeast surface and to clone 2C was measured by flowcytometry. As shown in FIG. 2, the native TCR on 2C is capable ofbinding QL9 variants that contain either tyrosine or histidine atposition 5 but not those containing glutamic acid. Each of the higheraffinity TCR mutants retained the ability to recognize the conservativetyrosine-substituted peptide, and they were likewise incapable ofrecognizing the glutamic acid-substituted peptide. However, several ofthe TCR mutants (qL2, qL5, and qL7) bound to the histidine-substitutedpeptide (albeit to different extents) whereas other mutants (qL 1, qL3,and qL8) did not bind this peptide (within the detection limits of thisassay). Thus, the CDR3α loop can influence the peptide fine specificityof recognition, but it is not the only region of the TCR involved. Theeffect on peptide specificity could be through direct interaction ofCDR3α residues with the variant peptide, as suggested from earlierstudies involving CDR3-directed selections [Sant' Angelo et al. (1996)Immunity 4:367-376; Jorgensen et al. (1992) Nature 355:224-230].Alternatively, binding energy may be directed at peptide-induced changesin the L^(d) molecule itself. The latter possibility is perhaps morelikely in the case of the 2C TCR:QL9/L^(d) interaction, as position 5 ofQL9 has been predicted to point toward the L^(d) groove [Schlueter etal. (1996) supra; Speir et al. (1998) Immunity 8:553-562]. Thefine-specificity analysis also shows that it is possible to engineer TCRwith increased, or at least altered, specificity for cognate peptides.Thus, directed evolution of only a short region (CDR3α) of a single TCRallows the isolation of many TCR variants with desirable peptide-bindingspecificities and/or increased binding affinities.

[0053] In order to determine the magnitude of the affinity increasesassociated with a selected CDR3α mutant, the wild type T7 scTCR and theqL2 scTCR were expressed as soluble forms in a yeast secretion system.Purified scTCR preparations were compared for their ability to block thebinding of a ¹²⁵I-labeled anti-L^(d) Fab fragments to QL9 or MCMV (SEQID NO:1) loaded onto L^(d) on the surface of T2-L^(d) cells [Manning(1998) supra; Sykulev et al. (1994) Immunity 1:15-22]. As expected,neither T7 nor qL2 scTCR were capable of inhibiting the binding of¹²⁵I-Fab fragments to T2-L^(d) cells upregulated with the MCMV (SEQ IDNO:1) peptide. However, both T7 and qL2 were capable of inhibiting thebinding of anti-L^(d) Fab fragments to QL9/L^(d) (FIG. 3). The qL2 scTCRvariant was as effective as unlabeled Fab fragments in inhibitingbinding, whereas the T7 scTCR was 160-fold less effective (average of140-fold difference among four independent titrations). The K_(D) valuesof the scTCR for the QL9/L^(d) were calculated from the inhibitioncurves to be 1.5 μM for T7 and 9.0 nM for qL2. The value for T7 is inclose agreement with the 3.2 μM K_(D) previously reported for the 2CscTCR [Manning et al. (1999) J. Exp. Med. 189:461-470]. These findingsshow that the yeast system, combined with CDR3α-directed mutagenesis,allows selection of mutants with at least about 100-fold higherintrinsic binding affinities for a particular pMHC ligand.

[0054] If the soluble scTCR has a high affinity for its pMHC ligand,then it is useful, like antibodies, as a specific probe for cell-surfacebound antigen. To confirm this, the soluble T7 and qL2 scTCR werebiotinylated, and the labeled-scTCR molecules were incubated withT2-L^(d) cells loaded with QL9, p2Ca, or MCMV (SEQ ID NO:1). The qL2scTCR, but not the T7 scTCR, yielded easily detectable staining of theT2 cells that had been incubated with QL9 or p2Ca (FIGS. 4A-4B). It issignificant that p2Ca-upregulated cells were also readily detected byqL2 scTCR, as p2Ca is the naturally processed form of the peptiderecognized by the alloreactive clone 2C and it has an even loweraffinity than the QL9/L^(d) complex for the 2C TCR [Sykulev et al.(1994) supra].

[0055] The high affinity receptors described in our study were derivedby variation at the VJ junction, the same process that operates veryeffectively in vivo through gene rearrangements in T cells (Davis andBjorkman (1988) Nature 334:395-402). The fact that we could readilyisolate a diverse set of high affinity TCR in vitro indicates that thereis not a genetic or structural limitation to high affinity receptors.This supports the view that inherently low affinities of TCRs found invivo are due to a lack of selection for higher affinity and perhaps aselection for lower affinity (Sykulev et al. (1995) Proc. Natl. Acad.Sci. USA 92:11990-11992; Valitutti et al. (1995) Nature 375:148-151;Rabinowitz et al. (1996) Proc. Natl. Acad. Sci. USA 93:1401-1405). Inthis respect, the higher affinity TCRs of the present invention nowprovide the reagents for directly testing hypotheses about the effectsof affinity on T cell responses (Davis et al. (1998) Ann. Rev. Immunol.16:523-544; Sykulev et al. (1995) supra; Valitutti et al. (1995) supra;Rabinowitz et al. 1996) supra).

[0056] In summary, we have shown that T cell receptors, which representa class of proteins as diverse as antibodies, can be engineered likeantibodies to yield high affinity, antigen-specific probes. Furthermore,a soluble version of the high affinity receptor can directly detectspecific peptide/MHC complexes on cells. Thus, these engineered proteinsare useful as diagnostics, for tumor cells, for example. Solublederivatives of the high affinity TCRs are useful or can be furtherengineered as high affinity, antigen-specific probes. The soluble TCRderivatives when appropriately labeled (or bound by a detectable ligandfor that soluble TCR) can serve as a probe for specific peptide/MCHCcomplexes on cells, for example, derived surfaces of tumor cells orother neoplastic cells, or antigens diagnostic of virus-infected cellsor other diseased cells. Other applications for high affinity TCR cellbound proteins or soluble derivatives include use in diagnosis or studyof certain autoimmune diseases. Where a characteristic peptide/MHC orother marker surface antigen is known or can be identified, a highaffinity, soluble TCR can be isolated for specific binding to that cellsurface moiety and used in diagnosis or in therapy. The high affinityTCR proteins, desirably the soluble derivatives, can be used bound tocytotoxic agents as therapeutics in cancer treatment or other disorderswhere cells to be desirably destroyed have a characteristic and specificcell surface moiety which is recognized by a high affinity TCR(desirably a soluble TCR protein). Similarly, a soluble high affinityTCR as described herein can be coupled to an imaging agent and used toidentify sites within the body where tumor cells reside where the TCRspecifically binds a tumor cell marker with high affinity andspecificity. A high affinity TCR bound to the surface of a cell ortissue which has been inappropriately targeted for autoimmunedestruction can reduce autoimmune tissue destruction by cytotoxiclymphocytes by competing with those cytotoxic lymphocytes for binding tothe cell surface of the targeted cells or tissue.

[0057] These results can also be considered in the context of animportant, basic question in T cell responses. Are the low affinitiespreviously observed for T cell receptors due to the absence of somaticmutations or due to in vivo selective pressures that act on the T cell?The high affinity receptors described in our study were derived byvariation at the VJ junction, the same process that operates veryeffectively in T cells [Davis and Bjorkman (1988) supra]. The fact thatwe could readily isolate a diverse set of high affinity TCR in vitroindicates that there is no structural or genetic limitation to highaffinity receptors. This supports the view that inherently lowaffinities of TCRs found in vivo are due to a lack of selection forhigher affinity and perhaps a selection for lower affinity [Sykulev etal. (1995) Proc. Natl. Acad. Sci. USA 92:11990-11992; Rabinowitz et al.(1996) supra]. In this respect, the higher affinity TCRs now provide thereagents for directly testing hypotheses about the effects of affinityon T cell responses [Davis et al. (1998) supra; Sykulev et al. (1995)supra; Valitutti et al. (1995) supra; Rabinowitz et al. (1996) supra].

[0058] It will be appreciated by those of skill in the art that, due tothe degeneracy of the genetic code, numerous functionally equivalentnucleotide sequences encode the same amino acid sequence.

[0059] Additionally, those of skill in the art, through standardmutagenesis techniques, in conjunction with the antigen-finding activityassays described herein, can obtain altered TCR sequences and test themfor the expression of polypeptides having particular binding activity.Useful mutagenesis techniques known in the art include, withoutlimitation, oligonucleotide-directed mutagenesis, region-specificmutagenesis, linker-scanning mutagenesis, and site-directed mutagenesisby PCR [see e.g. Sambrook et al. (1989) and Ausubel et al. (1999)].

[0060] In obtaining variant TCR coding sequences, those of ordinaryskill in the art will recognize that TCR-derived proteins may bemodified by certain amino acid substitutions, additions, deletions, andpost-translational modifications, without loss or reduction ofbiological activity. In particular, it is well-known that conservativeamino acid substitutions, that is, substitution of one amino acid foranother amino acid of similar size, charge, polarity and conformation,are unlikely to significantly alter protein function. The 20 standardamino acids that are the constituents of proteins can be broadlycategorized into four groups of conservative amino acids as follows: thenonpolar (hydrophobic) group includes alanine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan and valine; the polar(uncharged, neutral) group includes asparagine, cysteine, glutamine,glycine, serine, threonine and tyrosine; the positively charged (basic)group contains arginine, histidine and lysine; and the negativelycharged (acidic) group contains aspartic acid and glutamic acid.Substitution in a protein of one amino acid for another within the samegroup is unlikely to have an adverse effect on the biological activityof the protein.

[0061] Homology between nucleotide sequences can be determined by DNAhybridization analysis, wherein the stability of the double-stranded DNAhybrid is dependent on the extent of base pairing that occurs.Conditions of high temperature and/or low salt content reduce thestability of the hybrid, and can be varied to prevent annealing ofsequences having less than a selected degree of homology. For instance,for sequences with about 55% G-C content, hybridization and washconditions of 40-50° C., 6×SSC (sodium chloride/sodium citrate buffer)and 0.1% SDS (sodium dodecyl sulfate) indicate about 60-70% homology,hybridization and wash conditions of 50-65° C., 1×SSC and 0.1% SDSindicate about 82-97% homology, and hybridization and wash conditions of52° C., 0.1×SSC and 0.1% SDS indicate about 99-100% homology. A widerange of computer programs for comparing nucleotide and amino acidsequences (and measuring the degree of homology) are also available, anda list providing sources of both commercially available and freesoftware is found in Ausubel et al. (1999). Readily available sequencecomparison and multiple sequence alignment algorithms are, respectively,the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1997 )and ClustalW programs. BLAST is available on the Internet athttp://www.ncbi.nlm.nih.gov and a version of ClustalW is available athttp://www2.ebi.ac.uk.

[0062] Industrial strains of microorganisms (e.g., Aspergillus niger,Aspergillus ficuum, Aspergillus awamori, Aspergillus oryzae, Trichodermareesei, Mucor miehei, Kluyveromyces lactis, Pichia pastoris,Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis orBacillus licheniformis) or plant species (e.g., canola, soybean, corn,potato, barley, rye, wheat) may be used as host cells for therecombinant production of the TCR peptides. As the first step in theheterologous expression of a high affinity TCR protein or solubleprotein, an expression construct is assembled to include the TCR orsoluble TCR coding sequence and control sequences such as promoters,enhancers and terminators. Other sequences such as signal sequences andselectable markers may also be included. To achieve extracellularexpression of the scTCR, the expression construct may include asecretory signal sequence. The signal sequence is not included on theexpression construct if cytoplasmic expression is desired. The promoterand signal sequence are functional in the host cell and provide forexpression and secretion of the TCR or soluble TCR protein.Transcriptional terminators are included to ensure efficienttranscription. Ancillary sequences enhancing expression or proteinpurification may also be included in the expression construct.

[0063] Various promoters (transcriptional initiation regulatory region)may be used according to the invention. The selection of the appropriatepromoter is dependent upon the proposed expression host. Promoters fromheterologous sources may be used as long as they are functional in thechosen host.

[0064] Promoter selection is also dependent upon the desired efficiencyand level of peptide or protein production. Inducible promoters such astac are often employed in order to dramatically increase the level ofprotein expression in E. coli. Overexpression of proteins may be harmfulto the host cells. Consequently, host cell growth may be limited. Theuse of inducible promoter systems allows the host cells to be cultivatedto acceptable densities prior to induction of gene expression, therebyfacilitating higher product yields.

[0065] Various signal sequences may be used according to the invention.A signal sequence which is homologous to the TCR coding sequence may beused. Alternatively, a signal sequence which has been selected ordesigned for efficient secretion and processing in the expression hostmay also be used. For example, suitable signal sequence/host cell pairsinclude the B. subtilis sacB signal sequence for secretion in B.subtilis, and the Saccharomyces cerevisiae α-mating factor or P.pastoris acid phosphatase phoI signal sequences for P. pastorissecretion. The signal sequence may be joined directly through thesequence encoding the signal peptidase cleavage site to the proteincoding sequence, or through a short nucleotide bridge consisting ofusually fewer than ten codons, where the bridge ensures correct readingframe of the downstream TCR sequence.

[0066] Elements for enhancing transcription and translation have beenidentified for eukaryotic protein expression systems. For example,positioning the cauliflower mosaic virus (CaMV) promoter 1000 bp oneither side of a heterologous promoter may elevate transcriptionallevels by 10- to 400-fold in plant cells. The expression constructshould also include the appropriate translational initiation sequences.Modification of the expression construct to include a Kozak consensussequence for proper translational initiation may increase the level oftranslation by 10 fold.

[0067] A selective marker is often employed, which may be part of theexpression construct or separate from it (e.g., carried by theexpression vector), so that the marker may integrate at a site differentfrom the gene of interest. Examples include markers that conferresistance to antibiotics (e.g., bla confers resistance to ampicillinfor E. coli host cells, nptII confers kanamycin resistance to a widevariety of prokaryotic and eukaryotic cells) or that permit the host togrow on minimal medium (e.g., HIS4 enables P. pastoris or His⁻ S.cerevisiae to grow in the absence of histidine). The selectable markerhas its own transcriptional and translational initiation and terminationregulatory regions to allow for independent expression of the marker. Ifantibiotic resistance is employed as a marker, the concentration of theantibiotic for selection will vary depending upon the antibiotic,generally ranging from 10 to 600 μg of the antibiotic/mL of medium.

[0068] The expression construct is assembled by employing knownrecombinant DNA techniques (Sambrook et al., 1989; Ausubel et al.,1999). Restriction enzyme digestion and ligation are the basic stepsemployed to join two fragments of DNA. The ends of the DNA fragment mayrequire modification prior to ligation, and this may be accomplished byfilling in overhangs, deleting terminal portions of the fragment(s) withnucleases (e.g., ExoIII), site directed mutagenesis, or by adding newbase pairs by PCR. Polylinkers and adaptors may be employed tofacilitate joining of selected fragments. The expression construct istypically assembled in stages employing rounds of restriction, ligation,and transformation of E. coli. Numerous cloning vectors suitable forconstruction of the expression construct are known in the art (λZAP andpBLUESCRIPT SK-1, Stratagene, LaJolla, Calif., pET, Novagen Inc.,Madison, Wis. —cited in Ausubel et al., 1999) and the particular choiceis not critical to the invention. The selection of cloning vector willbe influenced by the gene transfer system selected for introduction ofthe expression construct into the host cell. At the end of each stage,the resulting construct may be analyzed by restriction, DNA sequence,hybridization and PCR analyses.

[0069] The expression construct may be transformed into the host as thecloning vector construct, either linear or circular, or may be removedfrom the cloning vector and used as is or introduced onto a deliveryvector. The delivery vector facilitates the introduction and maintenanceof the expression construct in the selected host cell type. Theexpression construct is introduced into the host cells by any of anumber of known gene transfer systems (e.g., natural competence,chemically mediated transformation, protoplast transformation,electroporation, biolistic transformation, transfection, or conjugation)(Ausubel et al., 1999; Sambrook et al., 1989). The gene transfer systemselected depends upon the host cells and vector systems used.

[0070] For instance, the expression construct can be introduced into S.cerevisiae cells by protoplast transformation or electroporation.Electroporation of S. cerevisiae is readily accomplished, and yieldstransformation efficiencies comparable to spheroplast transformation.

[0071] Monoclonal or polyclonal antibodies, preferably monoclonal,specifically reacting with a TCR protein at a site other than the ligandbinding site may be made by methods known in the art. See, e.g., Harlowand Lane (1988) Antibodies: A Laboratory Manual, Cold Spring HarborLaboratories; Goding (1986) Monoclonal Antibodies: Principles andPractice, 2d ed., Academic Press, New York; and Ausubel et al. (1999)Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NewYork.

[0072] High affinity TCR proteins in cell-bound or soluble form whichare specific for a particular pMHC are useful, for example, asdiagnostic probes for screening biological samples (such as cells,tissue samples, biopsy material, bodily fluids and the like) or fordetecting the presence of the cognate pMHC in a test sample. Frequently,the high affinity TCR proteins are labeled by joining, either covalentlyor noncovalently, a substance which provides a detectable signal.Suitable labels include but are not limited to radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent agents, chemiluminescentagents, magnetic particles and the like. Additionally the TCR proteincan be coupled to a ligand for a second binding molecules: for example,the TCR protein can be biotinylated. Detection of the TCR bound to atarget cell or molecule can then be effected by binding of a detectablestreptavidin (a streptavidin to which a fluorescent, radioactive,chemiluminescent, or other detectable molecule is attached or to whichan enzyme for which there is a chromophoric substrate available). U.S.patents describing the use of such labels and/or toxic compounds to becovalently bound to the scTCR protein include but are not limited toU.S. Pat. Nos. 3,817,837; 3,850,752; 3,927,193; 3,939,350; 3,996,345;4,277,437; 4,275,149; 4,331,647; 4,348,376; 4,361,544; 4,468,457;4,444,744; 4,640,561; 4,366,241; RE 35,500; 5,299,253; 5,101,827;5,059,413. Labeled TCR proteins can be detected using a monitoringdevice or method appropriate to the label used. Fluorescence microscopyor fluorescence activated cell sorting can be used where the label is afluorescent moiety, and where the label is a radionuclide, gammacounting, autoradiography or liquid scintillation counting, for example,can be used with the proviso that the method is appropriate to thesample being analyzed and the radionuclide used. In addition, there canbe secondary detection molecules or particle employed where there is adetectable molecule or particle which recognized the portion of the TCRprotein which is not part of the binding site for the cognate pMHCligand or other ligand in the absence of a MHC component as notedherein. The art knows useful compounds for diagnostic imaging in situ;see, e.g., U.S. Pat. Nos. 5,101,827; 5,059,413. Radionuclides useful fortherapy and/or imaging in vivo include ¹¹¹Indium, ⁹⁷Rubidium, ¹²⁵Iodine,¹³¹Iodine, ¹²³Iodine, ⁶⁷Gallium, ⁹⁹Technetium. Toxins include diphtheriatoxin, ricin and castor bean toxin, among others, with the proviso thatonce the TCR-toxin complex is bound to the cell, the toxic moiety isinternalized so that it can exert its cytotoxic effect. Immunotoxintechnology is well known to the art, and suitable toxic moleculesinclude, without limitation, chemotherapeutic drugs such as vindesine,antifolates, e.g. methotrexate, cisplatin, mitomycin, anthrocyclinessuch as daunomycin, daunorubicin or adriamycin, and cytotoxic proteinssuch as ribosome inactivating proteins (e.g., diphtheria toxin, pokeweedantiviral protein, abrin, ricin, pseudomonas exotoxin A or theirrecombinant derivatives. See, generally, e.g., Olsnes and Pihl (1982)Pharmac. Ther. 25:355-381 and Monoclonal Antibodies for Cancer Detectionand Therapy, Eds. Baldwin and Byers, pp. 159-179, Academic Press, 1985.

[0073] High affinity TCR proteins specific for a particular pMHC ligandare useful in diagnosing animals, including humans believed to besuffering from a disease associated with the particular pMHC. The sc TCRmolecules of the present invention are useful for detecting essentiallyany antigen, including but not limited to, those associated with aneoplastic condition, an abnormal protein, or an infection orinfestation with a bacterium, a fungus, a virus, a protozoan, a yeast, anematode or other parasite. The high affinity sc TCR proteins can alsobe used in the diagnosis of certain genetic disorders in which there isan abnormal protein produced. Exemplary applications for these highaffinity proteins is in the treatment of autoimmune diseases in whichthere is a known pMHC. Type I diabetes is relatively well characterizedwith respect to the autoantigens which attract immune destruction.Multiple sclerosis, celiac disease, inflammatory bowel disease, Crohn'sdisease and rheumatoid arthritis are additional candidate diseases forsuch application. High affinity TCR (soluble) proteins with bindingspecificity for the p/MHC complex on the surface of cells or tissueswhich are improperly targeted for autoimmune destruction can serve asantagonists of the autoimmune destruction by competing for binding tothe target cells by cytotoxic lymphocytes. By contrast, high affinityTCR proteins, desirably soluble single chain TCR proteins, whichspecifically bind to an antigen or to a p/MHC on the surface of a cellfor which destruction is beneficial, can be coupled to toxic compounds(e.g., toxins or radionuclides) so that binding to the target cellresults in subsequent binding and destruction by cytotoxic lymphocytes.The cell targeted for destruction can be a neoplastic cell (such as atumor cell), a cell infected with a virus, bacterium or protozoan orother disease-causing agent or parasite, or it can be a bacterium,yeast, fungus, protozoan or other undesirable cell. Such high affinitysc TCR proteins can be obtained by the methods described herein andsubsequently used for screening for a particular ligand of interest.

[0074] The high affinity TCR compositions can be formulated by any ofthe means known in the art. They can be typically prepared asinjectables, especially for intravenous, intraperitoneal or synovialadministration (with the route determined by the particular disease) oras formulations for intranasal or oral administration, either as liquidsolutions or suspensions. Solid forms suitable for solution in, orsuspension in, liquid prior to injection or other administration mayalso be prepared. The preparation may also, for example, be emulsified,or the protein(s)/peptide(s) encapsulated in liposomes.

[0075] The active ingredients are often mixed with excipients orcarriers which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients include but are not limited towater, saline, dextrose, glycerol, ethanol, or the like and combinationsthereof. The concentration of the high affinity TCR protein ininjectable, aerosol or nasal formulations is usually in the range of0.05 to 5 mg/ml. The selection of the particular effective dosages isknown and performed without undue experimentation by one of ordinaryskill in the art. Similar dosages can be administered to other mucosalsurfaces.

[0076] In addition, if desired, vaccines may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and/or adjuvants which enhance the effectiveness of the vaccine.Examples of adjuvants which may be effective include but are not limitedto: aluminum hydroxide; N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE); and RIBI, which contains threecomponents extracted from bacteria: monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. Such additional formulations and modes of administration asare known in the art may also be used.

[0077] The high affinity TCR proteins of the present invention and/orpMHC-binding fragments having primary structure similar (more than 90%identity) to the high affinity TCR proteins and which maintain the highaffinity for the cognate ligand may be formulated into vaccines asneutral or salt forms. Pharmaceutically acceptable salts include but arenot limited to the acid addition salts (formed with free amino groups ofthe peptide) which are formed with inorganic acids, e.g., hydrochloricacid or phosphoric acids; and organic acids, e.g., acetic, oxalic,tartaric, or maleic acid. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases, e.g., sodium, potassium, ammonium,calcium, or ferric hydroxides, and organic bases, e.g., isopropylamine,trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.

[0078] High affinity TCR proteins for therapeutic use, e.g., thoseconjugated to cytotoxic compounds are administered in a mannercompatible with the dosage formulation, and in such amount and manner asare prophylactically and/or therapeutically effective, according to whatis known to the art. The quantity to be administered, which is generallyin the range of about 100 to 20,000 μg of protein per dose, moregenerally in the range of about 1000 to 10,000 μg of protein per dose.Similar compositions can be administered in similar ways using labeledhigh affinity TCR proteins for use in imaging, for example, to detecttissue under autoimmune attack and containing the cognate pMHCs or todetect cancer cells bearing a cognate pMHC on their surfaces. Preciseamounts of the active ingredient required to be administered may dependon the judgment of the physician or veterinarian and may be peculiar toeach individual, but such a determination is within the skill of such apractitioner.

[0079] The vaccine or other immunogenic composition may be given in asingle dose; two dose schedule, for example two to eight weeks apart; ora multiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may include 1 to 10 or more separatedoses, followed by other doses administered at subsequent time intervalsas required to maintain and/or reinforce the immune response, e.g., at 1to 4 months for a second dose, and if needed, a subsequent dose(s) afterseveral months. Humans (or other animals) immunized with theretrovirus-like particles of the present invention are protected frominfection by the cognate retrovirus.

[0080] Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) Molecular Cloning, Second Edition, Cold SpringHarbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) MolecularCloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993)Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al.(eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.)Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oldand Primrose (1981) Principles of Gene Manipulation, University ofCalifornia Press, Berkeley; Schleif and Wensink (1982) Practical Methodsin Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic AcidHybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979)Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York. Abbreviations and nomenclature, where employed, are deemedstandard in the field and commonly used in professional journals such asthose cited herein.

[0081] All references cited in the present application are incorporatedby reference herein to supplement the disclosure and experimentalprocedures provided in the present Specification to the extent thatthere is no inconsistency with the present disclosure.

[0082] The following examples are provided for illustrative purposes,and are not intended to limit the scope of the invention as claimedherein. Any variations in the exemplified articles and/or methods whichoccur to the skilled artisan are intended to fall within the scope ofthe present invention.

EXAMPLES

[0083] General methods for making high affinity TCRs are given in U.S.patent application Ser. No. 09/009,388, filed Jan. 20, 1998, andWO99/36569, filed Jan. 20, 1999, which are hereby incorporated byreference to the extent not inconsistent with the disclosure herewith.

Example 1 Library Construction

[0084] The 2C scTCR used as the scaffold for directed evolution (T7)contained six mutations (βG 17E, βG42E βL81S, αL43P, αW82R, and αI118N)that have been shown to increase the stability of the TCR but stillallow pMHC binding [see, e.g., Shusta, E. V. et al. (1999) J. Mol. Biol.292, 949-956].

[0085] Mutagenic PCR of the T7 scTCR VαCDR3 was performed using anAGA2-specific upstream primer (GGCAGCCCCATAAACACACAGTAT (SEQ ID NO:3))and a degenerate downstream primer CTTTTGTGCCGGATCCAAATGTCAG(SNN)₅GCTCACAGCACAGAAGTACACGGCCGAGTCGCTC (SEQ IDNO:4). Underlined bases indicate the positions of silent mutationsintroducing unique BamHI and EagI restriction sites. The purified PCRproduct was digested with NdeI and BamHI and ligated to NdeI-BamHIdigested T7/pCT302 [Boder and Wittrup (1997) supra; Kieke et al. (1999)supra; Shusta et al. (1999) supra]. The ligation mixture was transformedinto DH10B electro-competent E. coli (Gibco BRI, Gaithersburg, Md.), andtransformants were pooled into 250 ml LB supplemented with ampicillin at100 μg/ml and grown overnight at 37° C. Plasmid DNA was transformed intothe yeast, (Saccharomyces cerevisiae) strain EBY 100 by the method ofGietz and Schiestl [Geitz et al. (1995) Yeast 11:355-360].

Example 2 Cell Sorting

[0086] The yeast library [Shusta et al. (1999) Curr. Opin. Biotechnol.10:117-122] was grown in SD-CAA (2% dextrose, 0.67% yeast nitrogen base,1% casamino acids (Difco, Livonia, Mich.)) at 30° C. to an OD₆₀₀=4.0. Toinduce surface scTCR expression, yeast were pelleted by centrifugation,resuspended to an OD₆₀₀=1.0 in SG-CAA (2% galactose, 0.67% yeastnitrogen base, 1% casamino acids), and incubated at 20° C. for about 24hr. In general, about 10⁷ cells/tube were incubated on ice for 1 hr with50 μl of QL9/L^(d)/IgG dimers [Dal Porto et al. (1993) supra] diluted inphosphate buffered saline, pH 7.4 supplemented with 0.5 mg/ml BSA(PBS-BSA). After incubation, cells were washed and labeled for 30 minwith FITC-conjugated goat anti-mouse IgG F(ab⁷)₂ (Kirkegaard & Perry,Gaithersburg, Md.) in PBS-BSA. Yeast were then washed and resuspended inPBS-BSA immediately prior to sorting. Cells exhibiting the highestfluorescence were isolated by FACS sorting with a Coulter 753 bench.After isolation, sorted cells were expanded in SD-CAA and induced inSG-CAA for subsequent rounds of selection. A total of four sequentialsorts were performed. The concentrations of QL9/L^(d)/IgG dimers usedfor staining were 50 μg/ml for sorts 1-3 and 0.5 μg/ml for the finalsort. The percentages of total cells isolated from each sort were 5.55,2.68, 2.56, and 0.58%, respectively. Aliquots of sorts 3 and 4 wereplated on SD-CAA to isolate individual clones which were analyzed byflow cytometry using a Coulter Epics XL instrument.

Example 3 Soluble scTCR Production

[0087] The T7 and qL2 open reading frames were excised from pCT302NheI-XhoI and ligated into NheI-XhoI digested pRSGALT, a yeastexpression plasmid [Shusta et al. (1999) supra]. Ligated plasmids weretransformed into DH10B electro-competent E. coli (Gibco BRL). PlasmidDNA was isolated from bacterial cultures and transformed intoSaccharomyces cerevisiae BJ5464 (αura3-52 trp1 leu2 1 his3 200pep4::HIS3prbl 1.6R canl GAL) [Shusta et al. (1999) supra]. Yeast clones weregrown in one liter SD-CAA/Trp (20 mg/L tryptophan) for 48 hr at 30° C.To induce scTCR secretion, cells were pelleted by centrifugation at4000×g, resuspended in one liter SG-CAA/Trp supplemented with 1 mg/mlBSA, and incubated for 72 hr at 20° C. Culture supernatants wereharvested by centrifugation at 4000×g, concentrated to about 50 ml, anddialyzed against PBS, pH 8.0. The 6His-tagged scTCRs were purified bynative nickel affinity chromatography (Ni-NTA Superflow, Qiagen,Valencia, Calif.; 5 mM and 20 mM imidazole, pH 8.0 wash; 250 mMimidazole elution) [Shusta et al. (1999) supra].

Example 4 Cell-binding Assays

[0088] The binding of soluble scTCRs to QL9/L^(d) was monitored in acompetition format as described previously [Manning et al. (1998) supra;Sykulev et al. (1994) supra]. Peptide-upregulated T2-L^(d) cells(3×10⁵/well) were incubated for one hour on ice in the presence of¹²⁵I-labeled anti-L^(d) Fabs (30-5-7) and various concentrations ofscTCRs. Bound and unbound ¹²⁵I 30-5-7 Fabs were separated bycentrifugation through olive oil/dibutyl phthalate. Inhibition curveswere constructed to determine inhibitor concentrations yielding 50% ofmaximal inhibition. Dissociation constants were calculated using theformula of Cheng and Pursoff [Cheng (1973) Biochem. Pharm.22:3099-3108]. To monitor direct binding of scTCRs to cell-bound pMHC,peptide-upregulated T2-L^(d) cells (5×10⁵/tube) were incubated for 40min on ice with biotinylated soluble scTCRs followed by staining for 30min with streptavidin-phycoerythrin (PharMingen, San Diego, Calif.).Cellular fluorescence was detected by flow cytometry.

Example 5 Identification of High Affinity TCRs that are Specific for aDifferent Peptide and a Different MHC Molecule (K^(b))

[0089] Using the same library of yeast-displayed mutants of the CDR3αregion of the TCR, it was possible to select for higher affinity TCRsthat are specific for yet a different peptide bound to a different MHCmolecule. In this case the peptide called SIYR (SIYRYYGL (SEQ ID NO:5))was bound to the MHC molecule called K^(b), and this ligand complex wasused in fluorescent form to select by flow cytometry. Sixteen clonesexpressing high affinity TCR were sequenced, each showing a differentsequence in the CDR3α region (Table 2).

[0090] As an example of the specificity of these TCRs, the mutant 3SQ2was stained with various agents, including the secondary reagent alone(SA:PE), the anti-Vβ antibody F23.2, and three peptide/K^(b) complexes(OVA/K^(b), dEV8/K^(b), and SIYR/K^(b)). As shown in FIG. 5, only thepMHC (SIYR/K^(b)) used in the original selection had sufficient affinityto bind to the mutant TCR. Wild-type TCR did not bind the SIYR/K^(b)ligand at any concentration tested (data not shown).

[0091] The mutant TCR 3SQ2 was also expressed as a soluble protein inthe yeast secretion system and tested after biotinylation for itsability to bind directly to pMHC on the surface of tumor cells. As shownin FIG. 6, the labeled 3SQ2 bound very well to tumor cells thatexpressed only the appropriate peptide SIYR. The staining was nearly asstrong as the high affinity anti-K^(b) monoclonal antibody B8.24.3, thatbinds to any K^(b) molecule (FIG. 6), regardless of the peptide present.

Example 6 Identification of High Affinity TCRs that are Specific for aDifferent Peptide Bound to the Same MHC Molecule (K^(b))

[0092] To determine if the same TCR scaffold could be used to isolatehigher affinity forms against yet a different peptide bound to the sameMHC, we screened the TCR CDR3α library with the peptide called dEV8(EQYKFYSV (SEQ ID NO:6)), bound to K^(b). After several sorts by flowcytometry with the biotinylated dEV₈/K^(b) ligand (followed byphycoerythrin-streptavidin, PE-SA), there was a significant enrichmentof yeast cells that bound to the dEV8/K^(b) (as indicated by PE levelsin FIG. 7).

[0093] Six of the clones that were isolated by selection with dEV8/K^(b)were sequenced and the CDR3 sequences all differed (Table 3). Thesesequences were similar in sequence, but different from, those isolatedby selection with SIYR/K^(b) (two examples, 3SQ2 and 3SQ5, are alsoshown in Table 3). It can be concluded that it is possible to isolatehigher affinity TCRs against different antigens, even using the same TCRlibrary of mutants.

[0094] To prove the antigen specificity of the isolated clones, one ofthe dEV₈/K^(b) selected clones (4d1) was examined with a panel ofdifferent antibodies and ligands (FIG. 6). As expected, this TCR reactedwith the three appropriate antibodies (anti-Vβ8 antibody F23.2, anti-HAtag antibody, and anti-His tag antibody) and the dEV8/K^(b) antigen, butnot with another antigen, OVA/K^(b). Wild type TCR did not bind toeither peptide/K^(b) complex (data not shown). Thus, the high affinityTCR was specific for the selected antigen.

Example 7 Identification of High Affinity TCRs by Creating a Random TCRLibrary in a Different Region of the TCR (complementarity-determiningregion 3 of the β chain)

[0095] The examples described above used a library of TCR that weremutated within a region of the α chain called CDR3. In order to showthat other regions of the TCR could also be mutated to yield higheraffinity TCR, a library of random mutants within five contiguous aminoacid residues of the CDR3 region of the β chain was produced, using theqL2 TCR mutant as the starting material. This library was then selectedwith the QL9/L^(d) ligand at concentrations below that detected with theqL2 mutant. Five yeast clones, selected by flow cytometry, weresequenced and each showed a different nucleotide and amino acid sequence(FIG. 8). There was remarkable conservation of sequence within the fiveamino acid region that was mutated, suggesting that this sequence motifhas been optimized for high affinity. We conclude that it is possible tomutate different regions of the TCR to yield derivatives having higheraffinity for a particular pMHC.

[0096] Although the description above contains many specificities, theseshould not be construed to limit the scope of the invention but asmerely providing illustrations of some of the presently-preferredembodiments of this invention. For example, ligands other than thosespecifically illustrated may be used. Thus the scope of the inventionshould be determined by the appended claims and their legal equivalents,rather than by the examples given. All references cited herein areincorporated to the extent not inconsistent with the disclosureherewith. TABLE 1 Structure and sequences of the 2C TCR CDR3α. Wild TypeTCR VαCDR3 2C ⁹³SGFASAL¹⁰⁴ (SEQ ID NO:7) Mutant Mutant TCR VαCDR3 TCRVαCDR3 q2r SSYGNYL (SEQ ID NO:8) q1r SLPPPLL (SEQ ID NO:17) q4r SRRGHAL(SEQ ID NO:9) q3r SIPTPSL (SEQ ID NO:18) q5r SSRGTAL (SEQ ID NO:10) qL6SNPPPLL (SEQ ID NO:19) q6r SHFGTRL (SEQ ID NO:11) qL7 SDPPPLL (SEQ IDNO:20) qL1 SMFGTRL (SEQ ID NO:12) qL8 SSPPPRL (SEQ ID NO:21) qL2 SHQGRYL(SEQ ID NO:13) qL10 SAPPPIL (SEQ ID NO:22) qL3 SYLGLRL (SEQ ID NO:14)qL4 SKHGIHL (SEQ ID NO:15) qL5 SLTGRYL (SEQ ID NO:16)

[0097] Alignment of amino acid sequences of mutant scTCRs isolated byyeast display and selection with QL9/L^(d). Display plasmids wereisolated from yeast clones after selection and sequenced to determineCDR3α sequences. Mutants m1, m2, m3, m4, m10 and m11 were isolated afterthe third round of sorting. All other mutants were isolated after thefourth round of sorting. TABLE 2 SIYR/K^(b) Binders (3SQ2,3SQ5): CDR3αSequences 3SQ5 SGTHPFL (SEQ ID NO:23) SK7 SGHLPFL (SEQ ID NO:24) K5rSDSKPFL (SEQ ID NO:25) K4r SSDRPYL (SEQ ID NO:26) SK8 SLERPYL (SEQ IDNO:27) SK2 SREAPYL (SEQ ID NO:28) K3r SLHRPAL*(SEQ ID NO:29) 3SQ2SLHRPAL*(SEQ ID NO:30) SK10 SSNRPAL (SEQ ID NO:31) K1r STDRPSL (SEQ IDNO:32) K2r SGSRPTL (SEQ ID NO:33) SK3 SLVTPAL (SEQ ID NO:34) SK1SATSPAL (SEQ ID NO:35) SK9 SSINPAL (SEQ ID NO:36) SK4 SASYPSL (SEQ IDNO:37) 3SQ1 SRWTSGL (SEQ ID NO:38) Consensus SGSRPAL (SEQ ID NO:39)

[0098] TABLE 3 Alignment of VαCDR3 Mutant Sequences with High Affinityfor dEVS/K^(b) (4d1, 4d2, 3Sd3, 3dS6, 3dS2, 3d2) and SIYR/K^(b) CloneCDR3α 4d1 SLTHHFL (SEQ ID NO:40) 4d2 SMTHHFL (SEQ ID NO:41) 3Sd3 SLSRPYL(SEQ ID NO:42) 3dS6 SLTRPYL (SEQ ID NO:43) 3dS2 STYRHYL (SEQ ID NO:44)3d2 SGLARPL (SEQ ID NO:45) 3SQ2 SLHRPAL (SEQ ID NO:46) 3SQ5 SGTHPFL (SEQID NO:47)

[0099] TABLE 4 Alignment of VβCDR3 Sequences of Mutant scTCRs Selectedfor High Affinity for QL9/L^(d) from a CDR3α Yeast Library Clone 95 9697 98 105 106 107 CDR3β WT 2C GGT GGG GGG GGC ACC TTG TAC GGGGTLY (SEQID NO:48) QB2 GGT GGG GGG GGG GTG TTG TAC GGGGVLY (SEQ ID NO:49) QB4 GGTTTG GGG GGG ATC CTC TAC GLGGILY (SEQ ID NO:50) QB6/8 GGT CAG GGC GGG GTGTTG TAC GQGGVLY (SEQ ID NO:51) QB7 GGT TCG GGG GGG ATC ATC TAC GSGGIIY(SEQ ID NO:52) QB9 GGT GGC GGG GGG ATC TTG TAC GGGGILY (SEQ ID NO:53)

[0100]

1 53 1 4 PRT ARTIFICIAL SEQUENCE misc_feature ()..() null peptide 1 MetCys Met Val 1 2 4 PRT ARTIFICIAL SEQUENCE misc_feature ()..() incubationpeptide 2 Ser Ile Tyr Arg 1 3 24 PRT ARTIFICIAL SEQUENCE misc_feature()..() upstream primer 3 Gly Gly Cys Ala Gly Cys Cys Cys Cys Ala Thr AlaAla Ala Cys Ala 1 5 10 15 Cys Ala Cys Ala Gly Thr Ala Thr 20 4 74 PRTARTIFICIAL SEQUENCE misc_feature ()..() downstream primer 4 Cys Thr ThrThr Thr Gly Thr Gly Cys Cys Gly Gly Ala Thr Cys Cys 1 5 10 15 Ala AlaAla Thr Gly Thr Cys Ala Gly Ser Asn Asn Ser Asn Asn Ser 20 25 30 Asn AsnSer Asn Asn Ser Asn Asn Gly Cys Thr Cys Ala Cys Ala Gly 35 40 45 Cys AlaCys Ala Gly Ala Ala Gly Thr Ala Cys Ala Cys Gly Gly Cys 50 55 60 Cys GlyAla Gly Thr Cys Gly Cys Thr Cys 65 70 5 8 PRT ARTIFICIAL SEQUENCEmisc_feature ()..() binding peptide 5 Ser Ile Tyr Arg Tyr Tyr Gly Leu 15 6 8 PRT ARTIFICIAL SEQUENCE misc_feature ()..() screening peptide 6Glu Gln Tyr Lys Phe Tyr Ser Val 1 5 7 7 PRT ARTIFICIAL SEQUENCEmisc_feature ()..() CDR3alpha sequence 7 Ser Gly Phe Ala Ser Ala Leu 1 58 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 8 SerSer Tyr Gly Asn Tyr Leu 1 5 9 7 PRT ARTIFICIAL SEQUENCE misc_feature()..() CDR3alpha sequence 9 Ser Arg Arg Gly His Ala Leu 1 5 10 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 10 Ser SerArg Gly Thr Ala Leu 1 5 11 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 11 Ser His Phe Gly Thr Arg Leu 1 5 12 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 12 Ser MetPhe Gly Thr Arg Leu 1 5 13 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 13 Ser His Gln Gly Arg Tyr Leu 1 5 14 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 14 Ser TyrLeu Gly Leu Arg Leu 1 5 15 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 15 Ser Lys His Gly Ile His Leu 1 5 16 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 16 Ser LeuThr Gly Arg Tyr Leu 1 5 17 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 17 Ser Leu Pro Pro Pro Leu Leu 1 5 18 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 18 Ser IlePro Thr Pro Ser Leu 1 5 19 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 19 Ser Asn Pro Pro Pro Leu Leu 1 5 20 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 20 Ser AspPro Pro Pro Leu Leu 1 5 21 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 21 Ser Ser Pro Pro Pro Arg Leu 1 5 22 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 22 Ser AlaPro Pro Pro Ile Leu 1 5 23 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 23 Ser Gly Thr His Pro Phe Leu 1 5 24 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 24 Ser GlyHis Leu Pro Phe Leu 1 5 25 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 25 Ser Asp Ser Lys Pro Phe Leu 1 5 26 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 26 Ser SerAsp Arg Pro Tyr Leu 1 5 27 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 27 Ser Leu Glu Arg Pro Tyr Leu 1 5 28 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 28 Ser ArgGlu Ala Pro Tyr Leu 1 5 29 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 29 Ser Leu His Arg Pro Ala Leu 1 5 30 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 30 Ser LeuHis Arg Pro Ala Leu 1 5 31 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 31 Ser Ser Asn Arg Pro Ala Leu 1 5 32 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 32 Ser ThrAsp Arg Pro Ser Leu 1 5 33 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 33 Ser Gly Ser Arg Pro Thr Leu 1 5 34 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 34 Ser LeuVal Thr Pro Ala Leu 1 5 35 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 35 Ser Ala Thr Ser Pro Ala Leu 1 5 36 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 36 Ser SerIle Asn Pro Ala Leu 1 5 37 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 37 Ser Ala Ser Tyr Pro Ser Leu 1 5 38 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 38 Ser ArgTrp Thr Ser Gly Leu 1 5 39 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 39 Ser Gly Ser Arg Pro Ala Leu 1 5 40 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 40 Ser LeuThr His His Phe Leu 1 5 41 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 41 Ser Met Thr His His Phe Leu 1 5 42 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 42 Ser LeuSer Arg Pro Tyr Leu 1 5 43 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 43 Ser Leu Thr Arg Pro Tyr Leu 1 5 44 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 44 Ser ThrTyr Arg His Tyr Leu 1 5 45 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 45 Ser Gly Leu Ala Arg Pro Leu 1 5 46 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3alpha sequence 46 Ser LeuHis Arg Pro Ala Leu 1 5 47 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3alpha sequence 47 Ser Gly Thr His Pro Phe Leu 1 5 48 7 PRTARTIFICIAL SEQUENCE misc_feature ()..() CDR3beta sequence 48 Gly Gly GlyGly Thr Leu Tyr 1 5 49 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..()CDR3beta sequence 49 Gly Gly Gly Gly Val Leu Tyr 1 5 50 7 PRT ARTIFICIALSEQUENCE misc_feature ()..() CDR3beta sequence 50 Gly Leu Gly Gly IleLeu Tyr 1 5 51 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..() CDR3betasequence 51 Gly Gln Gly Gly Val Leu Tyr 1 5 52 7 PRT ARTIFICIAL SEQUENCEmisc_feature ()..() CDR3beta sequence 52 Gly Ser Gly Gly Ile Ile Tyr 1 553 7 PRT ARTIFICIAL SEQUENCE misc_feature ()..() CDR3beta sequence 53Gly Gly Gly Gly Ile Leu Tyr 1 5

We claim:
 1. A method for using high affinity TCRs to identify ligandscomprising: labeling high affinity TCRs; contacting said labeled TCRswith ligands; identifying the ligand with which the labeled TCR isbound.
 2. The method of claim 1, wherein said label is selected from thegroup consisting of: fluorescent compounds, chemiluminescent compounds,radioisotopes and chromophores.
 3. The method of claim 1, wherein saidligands are peptide/MHC ligands.
 4. A method of using high affinity TCRsto bind to a selected peptide/MHC ligand comprising: labeling said highaffinity TCRs with a label that binds to the selected peptide/MHCligand; contacting said labeled high affinity TCRs with cells containingMHC molecules.
 5. The method of claim 4, wherein said label is selectedfrom the group consisting of: fluorescent compounds, chemiluminescentcompounds, radioisotopes and chromophores.
 6. A method for using highaffinity TCRs as diagnostic probes for specific peptide/MHC molecules onsurfaces of cells comprising: labeling high affinity TCRs with a labelthat binds to specific peptide/MHC ligands; contacting said TCRs withcells; detecting said label.
 7. A method for using high affinity TCRsthat bind to pMHCs for diagnostic tests comprising: labeling the highaffinity TCR with a detectable label; contacting said high affinity TCRwith cells; detecting the label.
 8. The method of claim 7, wherein thenumber of labels present is detected.
 9. The method of claim 7, whereinthe location of the labels is detected in an organism.
 10. The method ofclaim 7, wherein said label binds to specific peptide/MHC ligands,whereby cells that express specific peptide/MHC ligands are targeted.11. The method for blocking autoimmune destruction of cells comprising:contacting TCRs with high affinity for the site recognized by the Tlymphocytes on the surface of a target cell with cells, whereby theautoimmune destruction of cells is blocked.
 12. The method for usinghigh affinity TCRs to treat disease comprising: coupling a TCR having ahigh affinity for a neoplastic cell surface marker with a therapeuticcompound; and contacting said TCR with cells.
 13. A method of using highaffinity TCRs to inactivate pathogens comprising: binding a moleculewhich is toxic to the pathogen to the high affinity TCR; and contactingsaid TCR with cells that express said pathogen.
 14. The method of claim13, wherein said pathogen is selected from the group consisting of:virus, bacteria and protozoa.
 15. Soluble T cell receptors (TCRs) havinghigher affinity for a ligand than wild type TCRs.
 16. The soluble highaffinity TCRs of claim 15, wherein said ligand is a peptide/MHC ligand.17. The soluble high affinity TCRs of claim 15, wherein said highaffinity TCR is made by the method comprising: mutagenizing a TCR tocreate mutant TCR coding sequences; transforming DNA comprising themutant TCR coding sequences for mutant TCRs into yeast cells; inducingexpression of the mutant TCR coding sequences such that the mutant TCRsare displayed on the surface of yeast cells; contacting the yeast cellswith a fluorescent label which binds to the peptide/MHC ligand toproduce selected yeast cells; and isolating the yeast cells showing thehighest fluorescence.
 18. The soluble high affinity TCRs of claim 15isolated by yeast display.
 19. A DNA library comprising nucleic acidsencoding soluble high affinity TCRs, wherein said TCRs are made by themethod of mutagenizing a TCR to create mutant TCR coding sequences;transforming DNA comprising the mutant TCR coding sequences for mutantTCRs into yeast cells; inducing expression of the mutant TCR codingsequences such that the mutant TCRs are displayed on the surface ofyeast cells; contacting the yeast cells with a fluorescent label whichbinds to the peptide/MHC ligand to produce selected yeast cells; andisolating the yeast cells showing the highest fluorescence.
 19. Alibrary of T cell receptor proteins displayed on the surface of yeastcells which have higher affinity for the peptide/HC ligand than the wildtype T cell receptor protein, wherein said library is formed bymutagenizing a T cell receptor protein coding sequence to generate avariegated population of mutants of the T cell receptor protein codingsequence; transforming the T cell receptor mutant coding sequence intoyeast cells; inducing expression of the T cell receptor mutant codingsequence on the surface of yeast cells; and selecting those cellsexpressing T cell receptor mutants that have higher affinity for thepeptide/MHC ligand than the wild type T cell receptor protein.
 20. Amethod for cloning the gene for a high affinity TCR mutant into a systemthat allows expression of the mutant on the surface of T cellscomprising: mutating TCRs to create high affinity TCR mutants; cloningsaid TCR mutants into a vector; transfecting the vector into T cells;expressing the high affinity TCR mutant on the surface of T cells. 21.The method of claim 20, further comprising: selecting those T cells thatare activated by a peptide/MHC ligand more than the wild type.
 22. Themethod of claim 20, wherein the transfected/infected T cells are usedfor recognition of selected peptide-bearing MHC cells.
 23. T cells madeby the method of claim 20.